Tournefolic Acid B, Derived from Clinopodium chinense (Benth.) Kuntze, Protects Against Myocardial Ischemia/Reperfusion Injury by Inhibiting Endoplasmic Reticulum Stress-Regulated Apoptosis via PI3K/AKT Pathways

Keywords: Apoptosis, Endoplasmic reticulum stress, Myocardial ischemia/reperfusion injury, Oxidative stress, Tournefolic acid B

Abstract

Background:

Protecting the heart from ischemia/reperfusion (I/R) injury is a major research focus, as myocardial infarction remains a leading cause of death worldwide. Tournefolic acid B (TAB) is a novel compound derived from Clinopodium chinense (Benth.) Kuntze, a traditional Chinese herbal medicine known for its anti-inflammatory, hypoglycemic, antitumor, and antiradiation properties. However, the pharmacological effects of TAB have been rarely studied.

Purpose:

The PI3K/AKT pathway is crucial for regulating endoplasmic reticulum (ER) stress and apoptosis during I/R injury. This study investigates the cardioprotective effects of TAB against myocardial I/R injury and explores the underlying molecular mechanisms.

Study Design:

H9c2 cardiomyocytes were incubated with TAB for 24 hours and then exposed to hypoxia/reoxygenation (H/R). Isolated rat hearts were subjected to global ischemia and reperfusion with or without TAB treatment.

Methods:

Mechanistic studies were conducted in vitro and ex vivo using JC-1 staining, ROS detection, caspase activity assays, TUNEL staining, and Western blot analysis.

Results:

TAB significantly improved hemodynamic parameters (LVeDP, LVSP, +dP/dt_max, -dP/dt_min, and HR) in isolated rat hearts and reduced cardiomyocyte apoptosis. TAB inhibited oxidative stress by modulating antioxidant enzyme activities (SOD, CAT, GSH-Px). I/R injury triggered ER stress by activating proteins such as Grp78, ATF6, PERK, and eIF2α, all of which were suppressed by TAB. TAB enhanced phosphorylation of PI3K and AKT, inhibited CHOP and Caspase-12 expression, reduced JNK phosphorylation, and increased the Bcl-2/Bax ratio.

Conclusion:

TAB protects against myocardial I/R injury by suppressing PI3K/AKT-mediated ER stress, oxidative stress, and apoptosis, suggesting its promise as a therapeutic agent for ischemic cardiovascular diseases.

Introduction

Ischemic cardiovascular diseases are the leading cause of death due to their high morbidity and mortality. Restoring blood supply is critical for treatment, but reperfusion can paradoxically cause further myocardial I/R injury, contributing to myocardial stunning, arrhythmia, and cardiac failure. I/R injury also triggers ventricular remodeling and can result in heart failure or sudden infarction. Cardiomyocyte apoptosis is closely linked to I/R injury progression and prognosis. Protective effects of exogenous ER stress inhibitors have been demonstrated in I/R models.

During I/R injury, ATP depletion, oxidative stress, and calcium imbalance lead to accumulation of misfolded proteins in the ER, triggering the unfolded protein response (UPR) and ER stress. Under normal conditions, Grp78 binds ER transmembrane proteins ATF6, IRE1, and PERK, but ER stress causes Grp78 to release these proteins, activating them and promoting apoptosis. PI3K/AKT pathways are crucial for regulating ER stress and apoptosis. Clinopodium chinense is a traditional Chinese herb with various pharmacological effects, but the role of its component TAB in cardioprotection is not well understood. This study investigates TAB’s effects on I/R-induced toxicity in vitro and ex vivo, and elucidates the mechanisms involved.

Materials and Methods
Materials

TAB (≥98% purity) was extracted from Clinopodium chinense and identified by mass spectrometry. All cell culture materials were from Gibco. Assay kits for MDA, CK, CAT, LDH, GSH-Px, and SOD were from Jiancheng Bioengineering Institute. Antibodies for Western blotting were from Santa Cruz Biotechnology and Cell Signaling Technology.

Cell Culture and Hypoxia-Reoxygenation (H/R) Modeling

Rat H9c2 cardiomyocytes were cultured and exposed to hypoxia (6 h) using glucose-free DMEM in an anaerobic chamber, followed by reoxygenation (12 h) in high-glucose DMEM.

Cell Viability and Death Assays

Cell viability was assessed by MTT assay. LDH leakage was measured to evaluate cell death.

Caspase Activity Assays

Caspase-3 and caspase-9 activities were measured using fluorometric assay kits.

Intracellular ROS Measurement

ROS levels were detected using a total ROS detection kit and analyzed by flow cytometry.

Mitochondrial Membrane Potential

Changes in mitochondrial membrane potential (ΔΨm) were detected using JC-1 dye and fluorescence microscopy.

Animal Experiments

Adult male Sprague-Dawley rats were used. Hearts were excised and perfused in a Langendorff apparatus. I/R injury was induced by 45 min ischemia and 60 min reperfusion. TAB was added to perfusate at various concentrations before I/R. LY294002 (PI3K/AKT inhibitor) and verapamil (positive control) were used in comparison groups.

Histopathology

Hearts were fixed, sectioned, and stained with hematoxylin and eosin for histological analysis.

TUNEL Staining

Apoptosis was assessed by TUNEL staining and confocal microscopy.

Antioxidant Indices

Activities of SOD, CAT, and GSH-Px, and MDA levels were measured in cell and heart tissue homogenates.

Western Blotting

Protein expression of PI3K, AKT, ER stress markers, and apoptosis-related proteins was analyzed by Western blot.

Statistical Analysis

Data were analyzed by one-way ANOVA and Student-Newman-Keuls post hoc test. P < 0.05 was considered significant.

Results
TAB Attenuates H/R-Induced Cardiotoxicity and Caspase Activation in H9c2 Cells

Hypoxia and reoxygenation significantly reduced cell viability in H9c2 cells. TAB at low concentrations (3.125–50 μg/mL) had no cytotoxic effect, but pretreatment with TAB (1 μg/mL) significantly improved cell viability and reduced LDH release after H/R. TAB also suppressed H/R-induced activation of caspase-3 and caspase-9, confirming its anti-apoptotic effect.

TAB Prevents Cell Apoptosis and Enhances Antioxidant Enzyme Activities

H/R decreased mitochondrial membrane potential (ΔΨm), increased ROS and MDA, and reduced SOD, CAT, and GSH-Px activities. TAB pretreatment preserved ΔΨm, reduced ROS and MDA, and increased antioxidant enzyme activities, indicating protection against oxidative stress.

TAB Ameliorates I/R-Induced Heart Dysfunction in Isolated Rat Hearts

In the ex vivo Langendorff model, I/R decreased LVSP, +dP/dt_max, -dP/dt_min, and HR, and increased LVeDP. TAB treatment dose-dependently improved these parameters, with the highest dose restoring function close to control levels. Histopathology confirmed reduced myocardial damage with TAB. The PI3K/AKT inhibitor LY294002 abolished TAB’s protective effects, implicating the PI3K/AKT pathway.

TAB Reduces Myocardial Apoptosis and Oxidative Damage in Isolated Hearts

TUNEL staining showed increased apoptosis after I/R, which was significantly reduced by TAB. TAB also restored SOD, CAT, and GSH-Px activities and reduced MDA levels in heart tissue. These protective effects were diminished by LY294002.

Involvement of PI3K/AKT Pathway

TAB increased phosphorylation of PI3K and AKT and reduced the Bax/Bcl-2 ratio, indicating inhibition of apoptosis. These effects were confirmed in both heart tissue and H9c2 cells.

TAB Suppresses I/R-Induced ER Stress

I/R increased expression of ER stress markers Grp78, ATF6, phosphorylated PERK, CHOP, Caspase-12, and phosphorylated JNK. TAB treatment suppressed these markers and increased Bcl-2 expression, while reducing Bax, CHOP, Caspase-12, and P-JNK. LY294002 reversed these effects, confirming PI3K/AKT involvement.

Discussion

TAB, a polyphenolic compound from Clinopodium chinense, exerts significant cardioprotective effects against I/R injury in vitro and ex vivo. TAB reduces cell death, apoptosis, and oxidative stress, and improves heart function. Mechanistically, TAB inhibits ER stress and associated apoptosis by activating the PI3K/AKT pathway. TAB also enhances antioxidant defenses, further protecting cardiac cells. The PI3K/AKT pathway is central to TAB’s effects, as inhibition of this pathway abrogates its benefits. These findings highlight TAB as a promising candidate for developing new therapies for ischemic cardiovascular diseases.

Conclusion

TAB significantly protects against myocardial I/R injury by suppressing ER stress and oxidative stress through activation of the PI3K/AKT pathway, thereby inhibiting apoptosis. These results provide a theoretical basis for developing TAB as a novel therapeutic agent for ischemic cardiovascular diseases and YUM70 support the clinical application of Clinopodium chinense.